Difference gel electrophoresis using matched multiple dyes

ABSTRACT

A process and a kit are provided for detecting differences in two or more samples of protein. Protein extracts are prepared, for example, from each of a different group of cell samples to be compared. Each protein extract is labeled with a different one of a luminescent dye from a matched set of dyes. The matched dyes have generally the same ionic and pH characteristics but emit light at different wavelengths to exhibit a different color upon luminescence detection. The labeled protein extracts are mixed together and electrophoresed together. The gel is observed to detect proteins unique to one sample or present in a greater ratio in one sample than in the other. Those unique or excess proteins will fluoresce the color of one of the dyes used. Proteins common to each sample migrate together and fluoresce the same.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for detecting differences inthe protein composition of cells and cell extracts, and moreparticularly, to a process utilizing a matched pair of labeling reagentsfor detecting such differences.

2. Background of the Invention

Researchers studying various aspects of cell biology use a variety oftools to detect and monitor differences in cell structure, function anddevelopment. An essential part of studying cells is studying thedifferences and similarities in the protein composition between thedifferent cell types, stages of development and condition. Determiningdifferences in the protein content between normal and cancerous cells orwild type and mutant cells, for example, can be a valuable source ofinformation and a valuable diagnostic tool.

Mixtures of proteins can be separated into individual componentsaccording to differences in mass by electrophoresing in a polyacrylamidegel under denaturing conditions. One dimensional and two dimensional gelelectrophoresis have become standard tools for studying proteins. Onedimensional SDS (sodium dodecyl sulfate) electrophoresis through acylindrical or slab gel reveals only the major proteins present in asample tested. Two dimensional polyacrylamide gel electrophoresis (2DPAGE), which separates proteins by isoelectric focusing, i.e., bycharge, in one dimension and by size in the second dimension, is themore sensitive method of separation and will provide resolution of mostof the proteins in a sample.

The proteins migrate in one- or two-dimensional gels as bands or spots,respectively. The separated proteins are visualized by a variety ofmethods; by staining with a protein specific dye, by protein mediatedsilver precipitation, autoradiographic detection of radioactivelylabeled protein, and by covalent attachment of fluorescent compounds.The latter method has been heretofore only able to be performed afterthe isoelectric focusing step of 2D PAGE. Immediately following theelectrophoresis, the resulting gel patterns may be visualized by eye,photographically or by electronic image capture, for example, by using acooled charge-coupled device (CCD).

To compare samples of proteins from different cells or different stagesof cell development by conventional methods, each different sample ispresently run on separate lanes of a one dimensional gel or separate twodimensional gels. Comparison is by visual examination or electronicimaging, for example, by computer-aided image analysis of digitized oneor two dimensional gels.

Two dimensional electrophoresis is frequently used by researchers.O'Farrell, P. H., "High resolution two-dimensional electrophoresis ofproteins", Journal of Biological Chemistry, 250:4007-4021 (1975),separated proteins according to their respective isoelectric points inthe first dimension by the now well known technique of isoelectricfocusing and by molecular weight in the second dimension bydiscontinuous SDS electrophoresis. Garrels, J. I., "Two-dimensional GelElectrophoresis and Computer Analysis of Proteins Synthesized By ClonalCell Lines", Journal of Biological Chemistry, Vol. 254, No. 16,7961-7977 (1979), used a two dimensional gel electrophoresis system tostudy the pattern of protein synthesis in nerve cells and glial cells.Garrels conducted a comparative analysis of data from multiple samplesto correlate the presence of particular proteins with specificfunctions. Computerized scanning equipment was used to scan a section ofthe gel fluorogram, detect the spots and integrate their densities. Theinformation was stored and plotted according to intensity in each ofseveral different scans.

Urwin, V. E. and Jackson, P., "A multiple High-resolution MiniTwo-dimensional Polyacrylamide Gel Electrophoresis System: ImagingTwo-dimensional Gels Using A Cooled Charge-Coupled Device After StainingWith Silver Or Labeling With Fluorophore", Analytical Biochemistry195:30-37 (1991) describes a technique wherein several isoelectricfocusing (IEF) gels were used to separate proteins by charge, thenloaded onto a gradient slab gel such that the IEF gels were positionedend to end along the top of the slab gel. The gels were thenelectrophoresed. The resulting protein spots were visualized either bystaining the second dimensional slab gel with silver or by fluorescentlabeling following the isoelectric focusing step. Labeling must takeplace after the first electrophoresis, i.e., the isoelectric focusingbecause the presence of the fluorescein label on the protein changes theisoelectric point of the protein when subjected to electrophoresis. Inaddition, the label attaches to a sulfur on the protein forming anunstable bond which would tend to break during isoelectric focusing ifthe label is attached prior to the electrophoresis step. An article bySantaren, J. et al., "Identification of Drosophila Wing Imaginal DiscProteins by Two-Dimensional Gel Analysis and Microsequencing",Experimental Cell Research 206: 220-226 (1993), describes the use ofhigh resolution two dimensional gel electrophoresis to identify proteinsin Drosophila melanogaster. The dry gel was exposed to X-ray film forfive days. The developed X-ray film is analyzed by a computer todetermine the differences in the samples.

Two dimensional gel electrophoresis has been a powerful tool forresolving complex mixtures of proteins. The differences between theproteins, however, can be subtle. Imperfections in the gel can interferewith accurate observations. In order to minimize the imperfections, thegels provided in commercially available electrophoresis systems areprepared with exacting precision. Even with meticulous controls, no twogels are identical. The gels may differ one from the other in pHgradients or uniformity. In addition, the electrophoresis conditionsfrom one run to the next may be different. Computer software has beendeveloped for automated alignment of different gels. However, all of thesoftware packages are based on linear expansion or contraction of one orboth of the dimensions on two dimensional gels. The software cannotadjust for local distortions in the gels.

The object of the present invention is to eliminate the problemsassociated with gel distortions and to provide a simple, relatively fastand reliable method of comparing and contrasting the protein content ofdifferent samples.

BRIEF SUMMARY OF THE INVENTION

The foregoing objects have been achieved by the process of the presentinvention which provides a method of comparing multiple samples of cellextract. The method includes the steps of preparing samples of cellextract from each of at least two different groups of cells and exposingeach sample of extract to a different one of a set of matchedluminescent dyes to covalently bind the dye to the extract to label theextract. Each dye within the set of dyes is capable of covalentlybinding the extract, has generally the same ionic and pH characteristicsas all other dyes within the set of dyes and emits light at a wavelengthsufficiently different from all the other dyes within the set of dyes topresent different colored light. The method of the invention furtherincludes the steps of quenching the binding of the dye to the extract,mixing the samples of labeled extract to form a mixture,electrophoresing the mixture to separate the extract components anddetecting the difference in fluorescence intensity among the separatedlabeled components by luminescence detection.

In the preferred process of the present invention, the differences, ifany, between multiple samples of proteins extracted for example, fromdifferent cells, are detected by labeling each sample of such proteinswith a different one of a set of matched luminescent dyes. The matcheddyes have generally the same ionic and pH characteristics but absorband/or fluoresce light at different wavelengths, producing a differentcolor fluorescence. In addition, the dyes should be similar in size.After an incubation period sufficient to permit the formation ofcovalent bonds between the dye and a plurality of attachment sites onthe proteins in the cell extract, preferably reacting with up to about2% of the total available attachment sites, the free reactive dye isthen quenched to prevent further reaction with the proteins, the labeledsamples are then mixed together and co-electrophoresed on a single gel.The proteins common to each sample comigrate to the same position.Proteins which are different will migrate alone to different locationson the gel and will fluoresce different colors, thereby identifyingwhich initial sample has one or more proteins which differ from theother initial sample or samples.

The invention also includes a kit for performing the method of thepresent invention. The kit includes the matched set of dyes, and mayalso include a quench material for stopping the reaction between theprotein and the dye, and the electrophoresis gels.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of necessary fee.

FIG. 1 is a schematic diagram of the process of the present invention;

FIGS. 2a and 2b are images of proteins labeled with a preferred matchedpair of labels of the present invention run on a single SDSpolyacrylamide gel;

FIGS. 3 A, B, C and D are images of a portion of a two dimensional gelloaded with two different samples of bacterial extract, one IPTG-inducedand the other uninduced, each labeled with a different one of the dyesof the matched pair of dyes according to the process of the presentinvention; and,

FIGS. 4 A, B, C and D are images of a portion of a two dimensional gelloaded with two different samples of bacterial extract, one havingexogenously added carbonic anhydrase and one without carbonic anhydrase,each labeled with a different one of the dyes of the matched pair ofdyes according to the process of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The process of the present invention employs a matched set of dyeswherein each dye in the set is generally equal to the other dyes inionic and pH characteristics, and chemical reactivity for covalentattachment to proteins, yet fluoresces at a different wavelength,thereby exhibiting a different color luminescence when viewed. The dyesare preferably roughly equal in molecular weight, but need not be. Eachone of the dyes within the matched set of dyes is used to label proteinsin a different one of a set of different samples of cell extract so thateach cell extract sample is labeled with a different dye within the setof dyes. After labeling, the extracts are mixed and electrophoresed inthe same gel, either by one or two dimensional electrophoresis.

With reference to the schematic diagram of FIG. 1, a first cell extractis prepared by known techniques from a first group of cells, thenlabeled with the first dye of a matched pair of dyes. A second cellextract is prepared by known techniques from a second group of cellsthen labeled with the second dye of the matched pair of dyes. To labelthe cell extract mixture, the reactive form of the dye and the proteinextract are incubated for a period of time sufficient to allow for theformation of a covalent bond between the reactive form of the dye andpotential attachment or binding sites on the proteins in the extract.The period of time is generally from 15 to 30 minutes, depending on thetemperature. The temperature range is generally from about 0° C. to 25°C. The reaction between the dye and the proteins may be quenched after asufficient percentage of available binding sites on the protein moleculeare covalently bound to the dye. Any suitable known quenching materialmay be used.

The first and second group of cells can be any two sets of cells theprotein content of which one wishes to compare or contrast. For example,the first group of cells can be the wild-type, or normal, cells, and thesecond group of cells can be mutant cells from the same species.Alternatively, the first group of cells can be normal cells and thesecond group can be cancerous cells from the same individual. Cells fromthe same individual at different stages of development or differentphases of the cell cycle can be used also. The cells from a developingembryo, from the ventral furrow of Drosophila melanogaster, for example,can be harvested as the first group of cells and cells that developadjacent to the ventral furrow cells can be harvested as the secondgroup of cells. The differences in protein composition between cells ofthe same type from different species can also be the subject of study bythe process of the present invention. In addition, the process of thepresent invention can be used to monitor how cells respond to a varietyof stimuli or drugs. All of the events that might alter cell behavior asexpressed through protein changes can be detected without the need andexpense of high precision 2D PAGE systems. Those skilled in the art willrecognize that the proteins for comparison may also be derived frombiological fluids, such as serum, urine, or spinal fluid.

The labeled samples are mixed and, as illustrated in FIG. 1, applied inmeasured aliquots to one gel, then preferably subjected to 2D PAGE. Onedimensional SDS electrophoresis can be used instead of 2D PAGE. Theprocedures for running one dimensional and two dimensionalelectrophoresis are well known to those skilled in the art.

Proteins that the two cell groups have in common form coincident spots.The ratio of the fluorescent intensity between identical proteins fromeither group will be constant for the vast majority of proteins.Proteins that the two groups do not have in common will migrateindependently. Thus, a protein that is unique or of different relativeconcentration to one group will have a different ratio of fluorescenceintensity from the majority of protein spots, and will produce a colorspecific for one or the other of the protein extracts, depending on thelabel used. For example, the proteins that are in the first sample maybe labeled red, while the second group is labeled blue. Under conditionswhere exactly equal amounts of protein from each group is mixed togetherand run on the same gel the ratio of fluorescence intensity will be onefor the majority of proteins. Those proteins that are distinct to one orthe other group will have a fluorescence intensity ratio less than orgreater than one, depending on the order or ratioing.

The gel can be analyzed by a two wavelength fluorescence scanner, by afluorescent microscope or by any known means for detecting fluorescence.Gel analysis can be completely automated by means of computer aidedidentification of protein differences. Using an electronic detectionsystem such as a laser scanning system with a photo multiplier tube or acharged-coupled device (CCD) camera and a white light source, twoelectronic images are made of the wet gel using different known filtersets to accommodate the different spectral characteristics of thelabels. One image views fluorescence of the first dye using a firstfilter appropriate to filter out all light except that emitted at thewavelength of the first dye and the other image views fluorescence ofthe second dye using a second filter, appropriate to filter out alllight except that emitted at the wavelength of the second dye. Exposureis about 5 to 500 seconds. The differences in the samples can beidentified, either during electrophoresis or in less than 1/2 hourfollowing electrophoresis. Several software packages are commerciallyavailable which will either subtract the first image from the second toidentify spots that are different, or, alternatively, the images may bedivided to leave only the spots not common to both images. Insubtracting the images, like spots will cancel each other, leaving onlythose that are different. In ratio analysis, like spots will provide avalue of one. Differences will result in values greater than one lessthan one.

In conventional analysis, a control is run with known proteins for thecell type being studied. The known spots on the sample gel have to beidentified and marked, compared to the control and the second gel todetermine differences between the two gels. In the present invention,there is only one gel so no marking is necessary. In addition, thesoftware used on conventional processes for alignment of different gelsprior to comparing and contrasting protein differences does not correctfor local distortions and inconsistencies between two or more gels. Theprocess of the present invention eliminates the need for such correctionbecause the extracts for all samples to be tested are mixed and run onthe same gel. Any gel distortions are experienced equally by eachsample.

Selection and synthesis of the matched set of dyes is important. In theprocess of the present invention, the fluorescent dyes are covalentlycoupled to proteins, preferably via lysine residues of the proteins, butcoupling may also be to sulfhydryl or carboxylic acid groups in theproteins. Regulation of the pH of proteins to force attachment of labelsat one amino acid residue to the exclusion of other amino acids is awell known technique, as set forth in R. Baker, Organic Chemistry ofBiological Components, (Prentice Hall, pub. 1971). For analysis ofproteins, a plurality of attachment sites are labeled. The optimumpercentage of attachment sites labeled will depend on the dyes chosen.When the preferred dyes specifically discussed hereinbelow are used,preferably no more than 2% of the attachment sites and more preferably,slightly less than 1%, are labeled, to avoid rendering the proteininsoluble. Thus, where a typical protein is composed of about 7%lysines, there will be less than one modified amino acid per onethousand. A typical protein is composed of about 450 amino acids. Whenlysine is the attachment site, the covalent linkage destroys thepositive charge of the primary amine of the lysine. Because isoelectricfocusing depends on charge, it is important to compensate for the chargeloss. A basic residue should remain basic. Changing the pK_(a) of oneresidue per protein by as much as 3 can be tolerated, provided thebasicity or acidity of the modified residue, as the case may be, is notaltered. Dyes like rhodamine and fluorescein are not suitable because ofthe difference in charge.

The first group of dyes evaluated were the fluorescent cyanine dyesdescribed in Mujumdar, R. B. et al., "Cyanine dye labeling reagentscontaining isothiocyanate groups", Cytometry 10:11-19 (1989) andWaggoner et al., U.S. Pat. No. 5,268,486 entitled "Method for labelingand detecting materials employing arylsulfonate cyanine dyes" issued in1993 and incorporated herein by reference. The cyanine dyes have thefollowing general structure. ##STR1## where X and Y can be O, S or(CH₃)₂ --C, m is an integer from 1 to 3 and at least one of R₁, R₂, R₃,R₄, R₅, R₆ or R₇ is a reactive group which reacts with amino, hydroxy orsulfhydryl nucleophiles. The dotted lines represent the carbon atomsnecessary for the formation of one ring to three fused rings having 5 to6 atoms in each ring. R₃, R₄, R₆ and R₇ are attached to the rings. Thereactive moiety can be any known reactive group. Reactive groups thatmay be attached directly or indirectly to the chromophore to form R₁,R₂, R₃, R₄, R₅, R₆ or R₇ groups may include reactive moieties such asgroups containing isothiocyanate, isocyanate, monochlorotriazine,dichlorotriazine, mono- or di-halogen substituted pyridine, mono- ordi-halogen substituted diazine, maleimide, aziridine, sulfonyl halide,acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester,imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)-proprionamide, glyoxal and aldehyde.

The cyanine dyes described in the Waggoner et al. patent were thefluorophors of choice because of their intrinsic positive charge. Thecyanines attach to the protein via the activated ester of hexanoic acid.While the coupling destroys the charge of the lysine side chain, theintrinsic charge in the dye compensates. It in effect moves the chargeaway from the protein molecule but maintains the same overall chargewithin the sample to be electrophoresed. In the cyanine dye molecule,two functionalized indole rings are connected via a polyene linker. Thespectral characteristics of cyanine dyes can be easily modulated bysimply changing the length of the linker between the indole rings of thedye. A longer or shorter linker length will result in fluorescence atdifferent wavelengths and thus, different colors. However, changing thelength of the linker changes the molecular mass of the dye. Sinceelectrophoresis depends also on the mass of the proteins, the effect ofthe dye on a protein's mass can also be of concern. Because the proteinsare labeled before electrophoresing, the mass of the dye attached to theprotein must not significantly alter the relative differences in themolecular weights of the various proteins in the extracts. Molecularweight is not critical, however, because only a relatively small numberof sites on the protein are labeled. As indicated above, preferably lessthan 1%, up to about 2% of the possible attachment sites on the proteinsare labeled. If more are labeled, maintaining generally equal molecularweights for the dyes within the set of matched dyes becomes a greaterconcern.

The difference in molecular weight caused by changing the linker lengthin the fluorescent cyanine dyes can be compensated for by modulating thesize of an aliphatic chain R₁ or R₂, attached to one of the dye's indolerings. One of R₁ or R₂ must be a reactive group. These designconstraints led to the modification of the cyanines and the developmentof a dye of the general formula. ##STR2## wherein X and Y equal S, O, orCH₃ --C--CH₃, m is an integer from 1 to 3 and either R₁ or R₂ is areactive group capable of covalently binding to the protein in the cellextract, such as the reactive groups described above for the unmodifiedcyanine dyes. The dotted lines represent 1, 2 or 3 fused rings having 5or 6 carbon atoms in each ring. Each side should balance the other side.

An example of a matched pair of dyes developed according to the generalformula follows: ##STR3## (Propyl Cy-3-NHS) which fluoresces red and,##STR4## (Methyl Cy-5-NHS) which fluoresces far red in the spectrumwherein R is a reactive group. As stated above, O or S or a combinationthereof can be placed in the X and Y positions in place of (CH₃)₂ C--.

The cyanine dyes are one choice for the matched set of dyes of thepresent invention. Other dye compounds may be used in place of thecyanines, such as dipyrromethene boron difluoride dyes, the derivatized4,4-difluoro-4-bora-3a,4a,-diaza-S-indacene dyes, described in U.S. Pat.No. 4,774,339 to Haugland et al. and incorporated herein by reference,which are sold by Molecular Probes, Inc. under the trademark BODIPY®.The BODIPY® dyes, which have no net charge, are covalently linked tolysine side chains using an activated n-hydroxysuccinimidyl ester whichforms an amide bond. The result is the loss of the lysine positivecharge. Therefore, a positively charged linker group is used in thematched dyes of the invention to replace the lost primary amine with thelinker's tertiary amine. The procedures for making BODIPY® dyes aredescribed in U.S. Pat. No. 4,774,339. Addition of the positively chargedlinker is by techniques well known to those skilled in the art. A linkercan be designed with three functional groups; (1) to react with theBODIPY®-NHS ester, (2) to carry the desired charge, and (3) to beactivated so that the BODIPY®-linker construct will react with specificamino acid residues of the proteins in the extract.

The major considerations for the matched set of dyes are the maintenanceof charge and distinct and different spectral characteristics. Anyneutral dyes with a positive linker or any positively charged dyes,preferably each having a +1 charge, that otherwise satisfy therequirements described herein can serve as the dyes in the matched setof dyes of the present invention. Roughly equal molecular weight in thesamples of labeled protein is desirable, but as explained above, notcritical. The intrinsic positive charge of cyanine dyes isadvantageously used in the preferred embodiment to replace the positivecharge of lysine. The pK_(a) of cyanines and lysine are ratherdifferent; however, conditions were selected for dye:protein ratio to beless than one. This low level of labeling ensures that there will benegligible changes in the protein's migration on two-dimensionalelectrophoresis gels. Dyes may be used which match the pK_(a) of lysinemore closely. Alternately, dyes that modify other amino acid residuesmay be used, provided the amino acid's ionic characteristics arepreserved by the modification. Instead of a lysine, the attachment siteon the protein may be a sulfhydryl or carboxylic group. When asulfhydryl group is the attachment site on the protein, thecorresponding attachment site on the dye is an iodoalkyl group. When acarboxylic acid group is the attachment site on the protein, thecorresponding attachment site on the dye is a chloroketone or acarbodiimide.

It is anticipated that the method of the present invention also can beused to detect the presence of different nucleic acids in differentsamples. The charge of nucleic acids is very negative. The addition ofthe dye does not therefore alter the overall charge in nucleic acids sothe choice of the matched set of dyes does not have to compensate forcharge loss when nucleic acid analysis is contemplated. To facilitateattachment of the dye, nucleic acids can be modified to have a freeamino acid coming from the nucleic acid nucleus by techniques known tothose skilled in the art. A lysine would be suitable in this instancealso.

EXAMPLE 1

Synthesis of the dyes (Methyl Cy-5 and Propyl Cy-3):

1. Synthesis of indole derivatives (common to both dyes):

4.8 g (30 mmoles) of 2,3,3-trimethyl-(3H)-indole and 35 mmoles of thedesired bromoalkyl reagent (6-bromohexanoic acid or 1-bromopropane) in40 ml of 1,2-dichlorobenzene were heated to 110° C. under nitrogen gasand stirred overnight with refluxing. The product (acid indole, methylindole, or propyl indole) precipitated as an orangish gum. Thesupernatant was decanted and the gum was washed several times with ethylether. This intermediate was used as is.

2. CY-3 intermediate:

1.5 g (7.5 mmoles) of propyl indole was added to 1.6 g (7.6 mmoles) ofN-N' diphenyl formamidine in 20 ml glacial acetic acid and was refluxedfor 4 hrs. The solvent was removed under vacuum leaving a deep orangesyrup. This intermediate was used as is.

2a. Cy-5 intermediate:

The synthesis of the Cy-5 intermediate is the same as the synthesis ofthe Cy-3 intermediate in step 2 of the dye synthesis except that2-methylene-1,3,3-trimethylindoline was used instead of propyl indoleand the linker was malonaldehyde dianil. The gummy, bluish intermediatewas washed twice with ethyl ether.

3. Cy-3:

2.5 ml of triethylamine and 1.8 ml of anhydrous Ac₂ O were added to theintermediate from step 2., and the mixture was boiled for 5 minutes.1.70 g (5.0 mmoles) of acid indole was added and the mixture wasrefluxed for two hours. The solvent was removed under vacuum and theproducts were dissolved in 10 ml of EtOH.

3a. Cy-5:

The preparation of Cy-5 is the same as that of Cy-3 except that theintermediate from step 2a. was used instead of the intermediate fromstep 2.

4. Purification of the products from steps 3. and 3a.:

Methyl Cy-5 and propyl Cy-3 were separated from contaminating sideproducts by running flash chromatography with a silica gel solid phaseand 40% MeOH in dichloromethane as the mobile phase.

5. Activation of carboxyl groups:

The carboxylic acid moiety of each dye was converted into anN-hydroxysuccinimidyl ester by dissolving a quantity of purifiedmaterial in 5 ml of dry dimethylformamidine (DMF). 1.5 equivalents ofN-N' disuccinimidyl carbonate (DSC) was added with 0.1 ml drypyridine/100 mg dye. The reaction was refluxed at 60° C. for 90 minutesunder nitrogen.

EXAMPLE 2

Protein Labeling:

1. Bacterial culture:

Initial experiments were performed on E. coli that expressed thechimeric GAL4VP16 protein under the control of the lac promoter asdescribed in Chasman, D. I. et al., "Activation of yeast polymerase IItranscription by Herpesvirus VP16 and GAL4 derivatives in vitro",Molecular Cell Biology 9:4746-4749 (1989). Two cultures of bacteria weregrown to an OD₆₀₀ of 0.7 at 37° C. in 125 ml of standard LB mediumcontaining 50 μg/ml ampicillin. Isophenylthiogalactopyranoside (IPTG), anon-hydrolyzable analog of lactose, was added to one culture at a finalconcentration of 1 mM. Both cultures were incubated for an additional2.5 hours.

2. Protein isolation for two-dimensional gel electrophoresis:

Isolation of protein was as follows. The bacteria was isolated bycentrifugation. Each bacterial pellet was washed with sonication buffercontaining 5 mM Hepes KOH pH 8.4, 5 mM Mg(OA_(c))2. The pellet wasresuspended in sonication buffer containing 50 μg/ml RNase to a finalvolume of 100 μl. This was then sonicated in ice until the solution wasclear, usually several minutes. DNase was added to 50 μg/ml and thesample was incubated for 30 min at 0° C. Solid urea and CHAPS were addedto a final concentration of 8 M and 5% respectively. The sample wastaken off the ice and 1 volume of lysis buffer added. The sample waseither labeled immediately or stored at -80° C.

3. Protein labeling:

Propyl Cy-3-NHS was added to the first sample and Methyl Cy-5-NHS wasadded to the second sample of cell extract at a concentration of 2 nmoleof dye/50 μg of protein. The dye stock solution was typically 2 mM indimethyl formamide. The reaction was incubated at 0° C. for 30 minutes.Incubation times may vary from about 15 to about 30 minutes, dependingon the temperature and the type of cells being studied. Incubation canbe for 15 minutes when the temperature is about 25° C. The temperatureshould not be above that which will cause the proteins to be degraded.The labeled sample was immediately subjected to isoelectric focusing orstored at -80° C.

4. Protein isolation and labeling for SDS-gel electrophoresis:

Bacteria were grown and isolated by sonication as in step 2. of theprotein labeling procedure, except RNase or DNase was not added. Thecell extract was directly labeled as in step 3 of the protein labelingprocedure. SDS, glycerol, Tris HCl pH 6.8, and bromophenol blue wereadded to bring the final concentrations to 1%, 10%, 64 mM, and 5 μg/ml,respectively. The sample was then placed in a boiling water bath for 2minutes and then subjected to electrophoresis.

5. Determination of dye to protein ratio:

In order to prevent solubility problems with labeled proteins,conditions were chosen to only label 1-2% of the lysines in the cellextract. This is based on the assumption that 7% of an average protein'samino acids are lysine. The first step in determining the dye to proteinratio was the removal of free dye by adsorption to SM-2 beads (Bio-Rad).The protein concentration was determined by OD_(260/280). The dyecontent was determined by OD548 and OD650 for Propyl Cy-3 and MethylCy-5, respectively (ε=100,000 for both dyes).

EXAMPLE 3

Gel Electrophoresis:

1. Two-dimensional electrophoresis:

High resolution two-dimensional gel electrophoresis was carried out bywell known techniques.

2. SDS polyacrylamide gel electrophoresis:

SDS polyacrylamide gel electrophoresis was carried out by knowntechniques.

EXAMPLE 4

Fluorescence Gel Imaging:

At the end of electrophoresis, the gels were soaked in a solution of 25%methanol and 7% acetic acid. The fluorescently labeled proteins in thegel were imaged in the following manner. Gels were placed on a surfaceof anodized aluminum and irradiated at an incident angle of 60° with a300 W halogen lamp housed in a slide projector. The light exiting theprojector was passed through 1' diameter bandpass filters (ChromaTechnologies, Brattleboro Vt.), 545±10 nm and 635±15 nm for Cy-3 andCy-5, respectively. The images were collected on a cooled, CCD camera(Photometrics Inc., Tucson Ariz.) fitted with a 50 mm lens (NIKON) and adouble bandpass emission filter (Chroma Technologies, Brattleboro Vt.),587.5±17.5 nm and 695±30 nm for Cy-3 and Cy-5, respectively. The CCDcamera was controlled by a MACINTOSH II si computer running Photometricscamera controller software. Image integration time ranged from tenths ofseconds to several minutes. The excitation filters were housed in afilter wheel attached to the projector. Two successive images wererecorded with irradiation from the two filters without moving the gel.

EXAMPLE 5

Image processing:

The image files were transferred to a Personal Iris 4D/35 (SiliconGraphics Inc., Mountain View Calif.). The image files were thenprocessed using the DELTAVISION™ software (Applied Precision, MercerIsland Wash.). The two schemes were used to determine the differencesbetween the differently labeled samples on the gel:

1. Subtraction:

Each image can be considered as a grid-like array of pixel intensities.These arrays of values can be manipulated by a number of arithmeticoperations. Here one image was subtracted from the other. Because thetwo samples loaded onto the gel were not perfectly balanced for overallfluorescence, one image was multiplied by a balancing constant. Thisfactor was determined arbitrarily so that the number of differencesbetween the samples were kept small.

2. Ratio Imaging:

Here one image was divided by the other. Before this operation wasperformed the images were first normalized to a common intensity range.This was done by setting the minimum and maximum pixel values of eachimage to zero and an arbitrarily large value, 4095, the maximum possibleoutput value of the CCD camera employed. Intermediate pixel values werescaled linearly between these values. One image was then divided by theother. A balancing factor was also used here to keep the mean quotientat one. Regions of difference were those with a quotient greater thanone.

EXAMPLE 6

1. Difference SDS gel electrophoresis of induced GAL4VP16 expression inbacteria:

FIG. 2 shows images of Propyl Cy-3 and Methyl Cy-5 labeled proteins runon a single SDS polyacrylamide gel. Lanes 1-3 show Cy-3 labeled protein.The samples loaded in there lanes were:

Lane 1. Propyl Cy-3 labeled IPTG-induced bacterial extract.

Lane 2. Propyl Cy-3 labeled IPTG-induced bacterial extract plus MethylCy-5 labeled uninduced extract.

Lane 3. Propyl Cy-3 labeled purified GAL4VP16 protein.

Lanes 4-6 show Cy-5 labeled protein. The samples loaded in there laneswere:

Lane 4. Propyl Cy-3 labeled IPTG-induced bacterial extract.

Lane 5. Propyl Cy-3 labeled IPTG-induced bacterial extract plus MethylCy-5 labeled uninduced extract.

Lane 6 Propyl Cy-3 labeled purified GAL4VP16 protein.

Only Lane 5 showed Cy-5 fluorescence.

Lanes 7 and 8 show the subtracted product of Lane 2-Lane 5 and Lane3-Lane 6, respectively. The arrows point to the position of GAL4VP16 asconfirmed by the position of the purified GAL4VP16 band in lane 8. Theidentity of the upper bands is not known. However, there are severalproteins that are known to be induced by IPTG, includingβ-galactosidase.

Lanes 9-11 show Cy-5 labeled protein. The samples loaded in these laneswere:

Lane 9. Methyl Cy-5 labeled IPTG-induced bacterial extract.

Lane 10. Methyl Cy-5 labeled IPTG-induced bacterial extract plus PropylCy-3 labeled uninduced extract.

Lane 11. Methyl Cy-5 labeled purified GAL4VP16 protein.

Lanes 12-15 show Cy-5 labeled protein. The samples loaded in there laneswere:

Lane 12. Methyl Cy-5 labeled IPTG-induced bacterial extract.

Lane 13. Methyl Cy-5 labeled IPTG-induced bacterial extract plus PropylCy-3 labeled uninduced extract.

Lane 14. Methyl Cy-5 labeled purified GAL4VP16 protein. Only Lanes 12-15all showed some Cy-3 fluorescence. This is due to slight crossoverbetween the bandpass filters. This causes Cy-5 labeled material toappear when excited by Cy-3 light. The converse is not seen. Cy-3material is not visualized by Cy-5 excitation light. There are two waysto eliminate the crossover effects: design better bandpass filters orcomputationally remove the Cy-5 contribution to the Cy-3 image byknowing the crossover constant.

Lanes 15 and 16 show the subtracted product of Lane 10-Lane 13 and Lane11-Lane 14, respectively. The arrows point to the position of GAL4VP16as confirmed by the position of the purified GAL4VP16 band in Lane 16.The identity of the upper bands is not known. However, there are severalproteins that are known to be induced by IPTG, includingβ-galactosidase.

2. Difference two-dimensional gel electrophoresis of induced GAL4VP16expression in bacteria:

FIG. 3 shows images of a portion of a two-dimension gel loaded withPropyl Cy-3 labeled IPTG-induced bacterial extract plus Methyl Cy-5labeled uninduced extract.

Panel A. Images taken with Cy-3 excitation light showing theIPTG-induced proteins.

Panel B. Images taken with Cy-5 excitation light showing the uninducedproteins.

Panel C. Ratio of the Cy-3 image divided by the Cy-5 image.

Panel D. Overlay of the image in Panel C, colored red, and placed on topof the image from Panel B, colored blue.

3. Difference two-dimensional gel electrophoresis of bacteria extractwith exogenously added protein:

FIG. 4 shows images of a portion of a two-dimension gel loaded withPropyl Cy-3 labeled bacterial extract that had exogenously addedcarbonic anhydrase plus Methyl Cy-5 labeled extract without the addedcarbonic anhydrase.

Panel A. Image taken with Cy-3 excitation light showing the bacterialproteins plus carbonic anhydrase.

Panel B. Images taken with Cy-5 excitation light showing the bacterialproteins alone.

Panel C. Ratio of the Cy-3 image divided by the Cy-5 image.

Panel D. Overlay of the image in Panel C, colored red, and placed on topof the image from Panel B, colored blue.

The process of the present invention provides a simple and inexpensiveway to analyze the differences in protein content of different cells.The process eliminates problems which can occur using two separate gelswhich must be separately electrophoresed. The matched dyes used to labelthe different cell extracts allow simultaneous electrophoresis of two ormore different samples of cell extract in a single gel. While theinvention has been described with reference to two samples of cellextract and a matched pair of dyes, those skilled in the art willappreciate that more than two samples may be simultaneously tested usingan equal number of matched dyes. As long as the spectral characteristicsof the dyes can be manipulated to provide fluorescence at a number ofdifferent wavelengths resulting in visually distinct images and the pHand ionic characteristics of the dyes can be generally equalized tocompensate for changes made to the protein by virtue of covalent bondingto the dye, multiple dyes can be used.

What we claim is:
 1. A method of comparing protein composition ofinterest between at least two different cell samples comprising:(a)preparing an extract of proteins from each of said at least two cellsamples; (b) providing a set of matched luminescent dyes chosen fromdyes capable of covalently binding to proteins within said extract ofproteins, wherein each dye within said set(1) has a net charge whichwill maintain the overall net charge of the proteins upon such covalentbinding and has ionic and pH characteristics whereby relativeelectrophoretic migration of a protein labeled with any one of said dyesis the same as relative electrophoretic migration of said proteinlabeled with another dye in said set, (2) emits luminescent light at awavelength that is sufficiently different from the emitted luminescentlight of remaining dyes in said set to provide a detectably differentlight signal; (c) reacting each extract of proteins of step (a) with adifferent dye from said set of step (b) to provide dye-labeled proteins;(d) quenching the labeling reactions of step (c) between the dyes andthe protein; (e) mixing each of said dye labeled proteins to form asingle mixture of different dye-labeled protein; (f) electrophoresingthe mixture of step (e) by a predetermined electrophoresing methodcapable of separation within said mixture; and (g) detecting thedifference in luminescent intensity between the different dye-labeledprotein by luminescent detection.
 2. The method of claim 1 wherein saiddyes bind to a primary amine of a lysine residue of the protein and eachsaid dye within said luminescent dyes carries a net +1 charge.
 3. Themethod of claim 1 wherein said set of matched luminescent dyes arecyanine dyes having the following structure: ##STR5## wherein the dottedlines each represent carbon atoms necessary for the formation of one tothree fused rings having five to six atoms in each ring, X and Y areselected from the group consisting of S, O or CH₃ --C--CH₃, m is aninteger from 1 to 3, one of R₁ and R₂ is a reactive group and the otheris an alkyl.
 4. The method of claim 3 wherein said reactive group reactswith an amino in the protein and is selected from the group consistingof isothiocyanate, (CH₂)₄ COOH and (CH₂)₄ CON-hydroxysuccinimidyl ester.5. The method of claim 1 wherein said set of matched luminescent dyesare neutral dyes bound to the primary amino of a lysine residue in theprotein by a positively charged linker group.
 6. The method of claim 1wherein said set of matched luminescent dyes are derivatives ofdipyrromethene boron difluoride dyes having positively charged linkergroups to bind the dye to the primary amino of a lysine residue in theprotein.
 7. The method of claim 1 wherein said dyes covalently bind tolysines in the proteins.
 8. The method of claim 1 wherein said dyescovalently bind to sulfhydryl groups on the proteins.
 9. The method ofclaim 1 wherein said dyes covalently bind to carboxylic acid groups onthe proteins.
 10. The method of claim 1 wherein the step of detectingdifferences in luminescent intensity is by fluorescent microscopy. 11.The method of claim 1 wherein the step of detecting differences inluminescent intensity is by electronic imaging.
 12. The method of claim1 wherein the proteins have binding sites for covalent binding to saiddyes selected from the group consisting of lysine, carboxylic acid andsulfhydryl groups.
 13. The method of claim 1 wherein the proteinsbinding sites for covalent binding to said dyes, and said binding sitesare one of a moiety selected from the group consisting of lysine,carboxylic acid and sulfhydryl groups.
 14. A method of comparingproteins of interest between at least two different samplescomprising:(a) preparing a mixture of proteins from each of said atleast two samples; (b) providing a set of matched luminescent dyeschosen from dyes capable of covalently binding to said proteins withinsaid mixture of proteins, wherein each dye within said set(1) has ionicand pH characteristics whereby relative electrophoretic migration of aprotein labeled with any one of said dyes is the same as relativeelectrophoretic migration of said protein labeled with another dye insaid set, (2) emits luminescent light at a wavelength that issufficiently different from the emitted luminescent light of remainingdyes in said set to provide a detectably different light signal; (c)reacting each said mixtures of proteins of step (a) with a different dyefrom said set of step (b) to provide dye-labeled protein; (d) quenchingthe labeling reactions of step (c) between the dyes and the proteins;(e) mixing each of said dye labeled proteins to form a combined singlemixture of different dye-labeled proteins; (f) electrophoresing thecombined mixture of step (e) by a predetermined electrophoresing methodcapable of separating the dye-labeled proteins of interest within saidmixture; and (g) detecting the difference in luminescent intensitybetween the different dye-labeled proteins of interest by luminescentdetection.
 15. The method of claim 14 wherein said set of matchedluminescent dyes are cyanine dyes having the following structure:##STR6## wherein the dotted lines each represent carbon atoms necessaryfor the formation of one to three fused rings having five to six atomsin each ring, X and Y are each selected from the group consisting of O,S and CH₃ --C--CH₃, m is an integer from 1 to 3, one of R₁ and R₂ is areactive group and the other is an alkyl.
 16. The method of claim 15wherein said reactive group is selected from the group consisting of andisothiocyanate, (CH₂)₄ COOH and (CH₂)₄ CON-hydroxysuccinimidyl ester.17. The method of claim 14 wherein said set of matched luminescent dyesare derivatives of dipyrromethene boron difluoride dyes havingpositively charged linker groups to bind the dye to the extract.
 18. Amethod of comparing proteins of interest between at least two differentsamples comprising:(a) preparing a mixture of proteins from each of saidat least two samples; (b) providing a set of matched luminescent dyeschosen from a single class of dyes capable of covalently binding toproteins within said mixture of proteins, wherein each dye within saidset(1) has ionic and pH characteristics whereby relative electrophoreticmigration of a protein labeled with any one of said dyes is the same asrelative electrophoretic migration of said protein labeled with anotherdye in said set, (2) emits luminescent light at a wavelength that issufficiently different from the emitted luminescent light of remainingdyes in said set to provide a detectably different light signal; (c)reacting each mixture of proteins of step (a) with a different dye fromsaid set of step (b) to provide dye-labeled proteins; (d) quenching thelabeling reactions of step (c) between the dyes and the proteins; (e)mixing each of said dye labeled proteins to form a combined singlemixture of different dye-labeled proteins; (f) electrophoresing thecombined mixture of step (e) by a predetermined electrophoresing methodcapable of separating the dye-labeled proteins of interest within saidcombined mixture; and (g) detecting the difference in luminescentintensity between the different dye-labeled proteins of interest byluminescent detection.